The influence of CD26+ and CD26- fibroblasts on the regeneration of human dermo-epidermal skin substitutes.

CD26, also known as dipeptidyl peptidase IV (DPPIV), is a multifunctional transmembrane protein playing a significant role in the cutaneous wound healing processes in the mouse skin. However, only scarce data are available regarding the distribution and function of this protein in the human skin. Therefore, the aim of this study was to investigate the impact of CD26 deficiency in human primary fibroblasts on the regeneration of human tissue-engineered skin substitutes in vivo. Dermo-epidermal skin analogs, based on collagen type I hydrogels, were populated either with human CD26+ or CD26knockout fibroblasts and seeded with human epidermal keratinocytes. These skin substitutes were transplanted onto the back of immune-incompetent rodents. Three weeks post-transplantation, the grafts were excised and analyzed with respect to specific epidermal and dermal maturation markers. For the first time, we show here that the expression of CD26 protein in human dermis is age-dependent. Furthermore, we prove that CD26+ fibroblasts are more active in the production of extracellular matrix (ECM) both in vitro and in vivo and are necessary to achieve rapid epidermal and dermal homeostasis after transplantation.

Implant Fibrosis and the Underappreciated Role of Myofibroblasts in the Foreign Body Reaction.

Body implants and implantable medical devices have dramatically improved and prolonged the life of countless patients. However, our body repair mechanisms have evolved to isolate, reject, or destroy any object that is recognized as foreign to the organism and inevitably mounts a foreign body reaction (FBR). Depending on its severity and chronicity, the FBR can impair implant performance or create severe clinical complications that will require surgical removal and/or replacement of the faulty device. The number of review articles discussing the FBR seems to be proportional to the number of different implant materials and clinical applications and one wonders, what else is there to tell? We will here take the position of a fibrosis researcher (which, coincidentally, we are) to elaborate similarities and differences between the FBR, normal wound healing, and chronic healing conditions that result in the development of peri-implant fibrosis. After giving credit to macrophages in the inflammatory phase of the FBR, we will mainly focus on the activation of fibroblastic cells into matrix-producing and highly contractile myofibroblasts. While fibrosis has been discussed to be a consequence of the disturbed and chronic inflammatory milieu in the FBR, direct activation of myofibroblasts at the implant surface is less commonly considered. Thus, we will provide a perspective how physical properties of the implant surface control myofibroblast actions and accumulation of stiff scar tissue. Because formation of scar tissue at the surface and around implant materials is a major reason for device failure and extraction surgeries, providing implant surfaces with myofibroblast-suppressing features is a first step to enhance implant acceptance and functional lifetime. Alternative therapeutic targets are elements of the myofibroblast mechanotransduction and contractile machinery and we will end with a brief overview on such targets that are considered for the treatment of other organ fibroses.

A Near-Infrared-Triggered Dynamic Wrinkling Biointerface for Noninvasive Harvesting of Practical Cell Sheets.

Cell sheet engineering represents a new era of precise and efficient regenerative medicine, but its efficacy is limited by the elaborative preparation and the weak mechanics. Herein, a near-infrared (NIR)-triggered dynamic wrinkling biointerface was designed for rapid acquisition of practical cell sheets. The biocompatible NIR can initiate the photothermal-mechanical linkage cascade to efficiently dissolve the collagen supporting layer and release the high-quality cell sheets. The interfacial shear force generates with the dynamic wrinkling, playing an active role in accelerating the cell sheet release. High-quality and self-supporting cell sheets can be harvested within a few minutes, demonstrating a new paradigm of photothermal-mechanical manipulation. The transplantable cell sheets with outstanding physiological and mechanical performances were proven to promote wound healing in skin regeneration. This method may open a completely new front in thermal and mechanical responsive cascade to harvest cell sheets, facilitating their wide applications in regenerative medicine.

Histatin 1 enhanced the speed and quality of wound healing through regulating the behaviour of fibroblast.

OBJECTIVES: Histatin 1(Hst 1) has been proved to promote wound healing. However, there was no specific study on the regulation made by Hst 1 of fibroblasts in the process of wound healing. This research comprehensively studied the regulation of Hst 1 on the function of fibroblasts in the process of wound healing and preliminary mechanism about it. MATERIALS AND METHODS: The full-thickness skin wound model was made on the back of C57/BL6 mice. The wound healing, collagen deposition and fibroblast distribution were detected on days 3, 5 and 7 after injury. Fibroblast was cultured in vitro and stimulated with Hst 1, and then, their biological characteristics and functions were detected. RESULTS: Histatin 1 can effectively promote wound healing, improve collagen deposition during and after healing and increase the number and function of fibroblasts. After healing, the mechanical properties of the skin also improved. In vitro, the migration ability of fibroblasts stimulated by Hst 1 was significantly improved, and the fibroblasts transformed more into myofibroblasts, which improved the function of contraction and collagen secretion. In fibroblasts, mTOR signalling pathway can be activated by Hst 1. CONCLUSIONS: Histatin 1 can accelerate wound healing and improve the mechanical properties of healed skin by promoting the function of fibroblasts. The intermolecular mechanisms need to be further studied, and this study provides a direction about mTOR signalling pathway.

Can plantar fibroblast implantation protect amputees from skin injury? A recipe for skin augmentation.

Skin injuries remain a persistent problem for users of lower-limb prostheses despite sustained progress in prosthesis design. One factor limiting the prevention of skin injuries is that skin on the residual limb is not suited to bear the mechanical loads of ambulation. One part of the body that is suited to this task is the sole of the foot. Here, we propose a novel strategy to actively augment skin's tolerance to load, increasing its resistance to mechanically induced injuries. We hypothesise that the load tolerance of skin can be augmented by autologous transplantation of plantar fibroblasts into the residual limb dermis. We expect that introducing plantar fibroblasts will induce the overlying keratinocytes to express plantar-specific keratins leading to a tougher epidermis. Using a computational finite element model of a weight-bearing residual limb, we estimate that skin deformation (a key driver of pressure ulcer injuries) could be halved by reprogramming skin to a plantar-like phenotype. We believe this strategy could yield new progress in pressure ulcer prevention for amputees, facilitating rehabilitation and improving quality of life for patients.

The effects of oral mucosa-derived heterotopic fibroblasts on cutaneous wound healing.

An intriguing observation that has recently found support through clinical and experimental studies is that wounds of the oral mucosa tend to display faster healing and result in less scarring than in the skin. We aimed to investigate the potential of heterotopic oral mucosal fibroblasts in cutaneous wounds while determining the main differences between wounds conditioned with either the oral mucosa or dermis-derived human fibroblasts. A total of 48 nude mice were divided into four groups: control, sham, dermal fibroblast (DF), and oral fibroblast (OF). Fibroblasts were isolated, cultured, and seeded onto fibrin scaffolds for transfer to full-thickness dorsal wounds. Cell viability, wound area, healing rate, vascularization, cellular proliferation, dermal thickness, collagen architecture, and subtypes were evaluated. Both cell groups had a viability of 95% in fibrin gel prior to transfer. None of the wounds fully epithelialized on day 10, while all were epithelialized by day 21, which resulted in scars of different sizes and quality. Healing rate and scars were similar between the control and sham groups, whereas fastest healing and least scarring were noted in the OF group. Dermal thickness was highest in the DF group, which was also supported by highest levels of collagen types I and III. Proliferative cells and vascular density were highest in the OF group. DF result in healing through a thick dermal component, while oral fibroblasts result in faster healing and less scarring through potentially privileged angiogenic and regenerative gene expression.

In Vivo Activity of Genetically Modified Cells Preseeded in Rat Vascularized Composite Allografts.

OBJECTIVE: Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model. RESULTS: Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P = .02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs-an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes.

Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Exosomes derived from autologous dermal fibroblasts promote diabetic cutaneous wound healing through the Akt/ß-catenin pathway.

Diabetic cutaneous wounds are one of the complications of diabetes mellitus (DM) and are difficult to cure at present. Autologous dermal fibroblasts (DFs) have shown great promise in skin regeneration and repair. However, whether exosomes derived from autologous dermal fibroblasts (DF-Ex) can be used to accelerate diabetic cutaneous wound healing is unclear. In this study, human umbilical vein endothelial cells (HUVECs) were treated with high glucose. We found that DF-Ex could reverse the damage produced by high glucose in HUVECs in vitro. A high-fat diet and streptozotocin were used to establish a rat model of type 2 diabetes mellitus (T2DM), and a diabetic cutaneous wound model was established in the T2DM rats. We discovered that subcutaneous injections of DF-Ex could significantly promote re-epithelialization, collagen deposition, skin cell proliferation, angiogenesis and inhibit inflammation to accelerate diabetic cutaneous wound healing. We further explored the underlying mechanism and found that DF-Ex exerted positive effects by activating the Akt/ß-catenin pathway. This research revealed that DF-Ex may provide a new treatment strategy for diabetic cutaneous wound healing.

Global Gene Expression of Cultured Human Dermal Fibroblasts: Focus on Cell Cycle and Proliferation Status in Improving the Condition of Face Skin.

Chronological skin ageing is an inevitable physiological process that results in thin and sagging skin, fine wrinkles, and gradual dermal atrophy. The main therapeutic approaches to soft tissue augmentation involve using dermal fillers, where natural fillers, such as autologous fibroblasts, are involved in generating dermal matrix proteins. The aim of this study was to determine the global transcriptome profile of three passages of dermal autologous fibroblasts from a male volunteer, focusing on the processes of the cell cycle and cell proliferation status to estimate the optimal passage of the tested cells with respect to their reimplantation. We performed K-means clustering and validation of the expression of the selected mRNA by qRT-PCR. Ten genes were selected (ANLN, BUB1, CDC20, CCNA2, DLGAP5, MKI67, PLK1, PRC1, SPAG5, and TPX2) from the top five processes annotated to cluster 5. Detailed microarray analysis of the fibroblast genes indicated that the cell population of the third passage exhibited the highest number of upregulated genes involved in the cell cycle and cell proliferation. In all cases, the results of qRT-PCR confirmed the differences in expression of the selected mRNAs between fibroblasts from the primary culture (C0) and from the first (C1), second (C2), and third (C3) cell passage. Our results thus suggest that these cells might be useful for increasing fibroblast numbers after reimplantation into a recipient's skin, and the method used in this study seems to be an excellent tool for autologous transplantation allowing the rejuvenation of aging skin.

Mitochondria-specific delivery system for targeted regulation of mitochondrial gene expression.

Targeted regulation of mitochondrial gene expression is challenging due to the lack of a mitochondria-specific delivery system. We have previously developed various stimuli-responsive nanoparticle (NP)-based delivery systems to transport nucleic acids for regulation of target gene expression. This protocol describes the design and preparation of an NP platform for mitochondria-specific gene delivery (mito-NP). We use mito-NP in primary liver fibroblasts that are transplanted into mice. Mito-NP can be used to deliver various nucleic acid therapeutics and to treat mitochondria-regulated diseases. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Combination therapy of trichloroacetic acid, human autologous fibroblast injection and fibroblast seeded microfibrous collagen scaffold as a novel treatment for osteomyelitis diabetic foot ulcer.

A severe complication associated with diabetes is diabetic foot ulcer (DFU). Most patients with DFU require amputation. Although treatment of non-healing diabetic ulcers is challenging, the use of novel therapies can be effective. In this report, we present the case of a woman with type 2 diabetes with DFU-related osteomyelitis, who was treated with a combination therapy of trichloroacetic acid, calcium alginate and foam dressings, human autologous fibroblast injection, and a fibroblast cell-seeded collagen scaffold. The results showed the positive effects of combination therapy on DFU. In the initial treatment, the wound area was measured to be 14 × 7 cm2 , with a depth of 4 cm. After 6 months, the wound was measured to be 1.5 cm2 , showing a 90% reduction of the wound area. Overall, this combination therapy was highly effective in the treatment of DFU-related osteomyelitis, and could markedly prevent amputation among DFU patients.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.

Comparison of similar cells: Mesenchymal stromal cells and fibroblasts.

Almost from all organs, both mesenchymal stromal cells and fibroblasts can be isolated. Mesenchymal stromal cells (MSCs) are the most preferred cellular therapeutic agents with the regenerative potential, and fibroblasts are one of the most abundant cell types with the ability to maintain homeostasis. Because of the promising properties of MSCs, they have been well studied and their differentiation potentials, immunomodulatory potentials, gene expression profiles are identified. It has been observed that fibroblasts and mesenchymal stromal cells have similar morphology, gene expression patterns, surface markers, proliferation, differentiation, and immunomodulatory capacities. Thus, it is hard to distinguish these two cell types. Epigenetic signatures, i.e., methylation patterns of cells, are the only usable promising difference between them. Such significant similarities show that these two cells may be related to each other.

Searching for Promoters to Drive Stable and Long-Term Transgene Expression in Fibroblasts for Syngeneic Mouse Tumor Models.

Tumor is a complex system of interactions between cancer cells and other cells of the tumor microenvironment. The cancer-associated fibroblasts (CAFs) of the tumor microenvironment remain in close contact with the cancer cells and play an important role in cancer progression. Genetically, CAFs are more stable than cancer cells, making them an attractive target for genetic modification in gene therapy. However, the efficiency of various promoters for transgene expression in fibroblasts is scarcely studied. We performed a comparative analysis of transgene long-term expression under the control of strong cytomegalovirus promoter (pCMV), constitutive cell promoter of the PCNA gene (pPCNA), and the potentially fibroblast-specific promoter of the IGFBP2 gene (pIGFBP2). In vitro expression of the transgene under the control of pCMV in fibroblasts was decreased soon after transduction, whereas the expression was more stable under the control of pIGFBP2 and pPCNA. The efficiency of transgene expression was higher under pPCNA than that under pIGFBP2. Additionally, in a mouse model, pPCNA provided more stable and increased transgene expression in fibroblasts as compared to that under pCMV. We conclude that PCNA promoter is the most efficient for long-term expression of transgenes in fibroblasts both in vitro and in vivo.

Does survivin overexpression enhance the efficiency of fibroblast cell-based wound therapy?

Cell-based wound therapy is faced with some limiting factors that decrease the therapeutic efficacy of transplanted cells. In this study, we aimed to genetically modify fibroblast cells with anti-apoptotic Survivin gene (Birc5) before cell transplantation. In vitro, pIRES2-eGFP-Survivin plasmid was transfected into the fibroblast cells and the growth curve was evaluated for transfected and normal cells performing MTT assay. In vivo, two 6-diameter cutaneous wounds were created at mice dorsal skin. Fibrin clot was used as a delivery vehicle to transfer cells into the wound bed. The effects of four treatment groups including (a) Cell-SVV-Clot (b) Cell-GFP-Clot, (c) Normal cell-Clot and, (d) Clot alone were evaluated. After 1,2,3,7 and 14 days post-transplantation, the wounds were photographed for evaluating the wound closure rate and wound samples were obtained. Angiogenesis and formation of granulated tissue were assessed via H&E staining for wound samples. The expression levels of Survivin, VEGF, and bFGF genes were also determined using qRT-PCR. The MTT assay showed similar proliferation potential of transfected cells with normal cells verifying that Survivin had no detrimental effect. Compared to the Normal cell-Clot group, the Survivin overexpression was seen for 3 days in the Cell-SVV-Clot group verifying the cell survival during the early stage of wound healing. The Survivin further upregulated VEGF and bFGF expressions resulting in more angiogenesis and formation of granulated tissue by day 3 and 14. The treated wounds with Cell-SVV-Clot were regenerated with a higher wound closure rate by day 7 compared to Normal cell-Clot and Clot groups. Survivin enhanced wound healing through induction of VEGF and bFGF at particular times post-wounding that led to a more structured-epidermis with higher angiogenesis and granulation tissue formation rate.

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency.

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.

The Causes for the Loss of Cellular Material at the Qualification Stage and the Influence of the Patient's Flora on Culture Infection.

There are several causes leading to the loss of cellular material earmarked for transplantation. This paper aims to evaluate the number of lost donors and lost cells in the culture by means of verifying both the results of the qualification tests and the presence of microorganisms in the cellular material. The analysis involved 86 donors hospitalized for thermal burns, from whom cells were harvested for keratinocyte and fibroblast cultures in the years 2011 to 2015. All potential donors underwent qualification tests: Anti-HIV-1,2; HBsAg; Anti-HCV-Ab; HBc, and a specific test for syphilis. In the case of skin fragments collected for culturing, the microbiological tests included the carrying fluid, the medium after 1 change, and the medium during culturing and before transplantation. Skin donors for cell cultures were assigned to the groups based on if the skin was collected up to 7 days following the burn or later. On average, 12% of the disqualifications were reported among donors for cell culturing. The most frequent cause of donor disqualification (54% of all disqualifications) was a positive HBc(+). The occurrence of fungal infections detected in the cellular material was over 30%. Establishing the culture after day 7 following the injury immediately increases the risk of infection by 25% in comparison to those cultures established before or on day 7 following the injury. Proper disinfection of donor place is crucial, but sometimes insufficient for maintenance sterility in cell culture. The risk of infection increases 25% after 7 following the injury.